Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus

Abstract

Objective: This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus.

Methods: Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively.

Results: EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%.

Conclusion: Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.

Keywords: EgG1Y162 antigen; fluorescent quantitative PCR; phylogenetic tree; prokaryotic expression.

Figure 1

Analysis of EgG1Y162 gene and protein expression. A. Relative expression level of egG1Y162 gene in developmental stages of Echinococcus granulosus. Expression of egG1Y162 gene was analyzed by fluorescent quantitative RT-PCR. Housekeeping gene egAct II was used as an internal control. B. Results of EgG1Y162 recombinant protein expression by SDS-PAGE electrophoresis analysis. Expression of pET-41a/EgG1Y162 plasmid was induced by IPTG (final concentration of 0.5 mmol/L) for 0 h, 2 h, 4 h, 6 h, respectively, at 30°C. C. Results of purified EgG1Y162 recombinant protein by SDS-PAGE electrophoresis analysis. EgG1Y162 recombinant protein was eluted with imidazole at concentrations of 0, 100, 200, 300, 500 mmol/L (pH 7.4).

Figure 2

Antigenicity analysis of EgG1Y162 protein. Antigenicity of EgG1Y162 protein was analyzed by Western Blot analysis. Serum samples from Echinococcus infected human/dog and from normal human/dog were used. A. Representative Western Blot results of dog serum blotting with EgG1Y162 protein. Lane 1: Protein molecular weight marker; Lane 2-7: serum samples from Echinococcus infected dog; Lane 8-13: serum samples from normal dog. B. Representative Western Blot results of Echinococcus infected human serum blotting with EgG1Y162 protein. Lane 1: protein molecular weight marker; Lane 2-12: serum samples from Echinococcus infected human. C. Representative Western Blot results of normal human serum blotting with EgG1Y162 protein. Lane 1: protein molecular weight marker; Lane 2-11: serum samples of normal human.

Figure 3

Analysis of gene structure. Gene constructions of eg95, egG1Y162 and emy162 were analyzed by online Spidey (http://www.ncbi.nih.gov/spidey). Gray rectangles represent exons and dark grey bold lines represent introns.

Figure 4

Amino acid sequence alignment of EgG1Y162, EMY162, EG95 and EM95. BLAST analysis was performed for amino acid sequence alignment. Strictly conserved residues are highlighted in bright yellow and partially conserved residues are depicted in bright green.

Figure 5

Phylogenetic analysis of EgG1Y162 protein. MEGA4 software was used for analyzing the evolutionary relationship of EgG1Y162 protein. And the phylogenetic tree of EgG1Y162 protein was depicted. Numbers indicate the phylogenetic relationship.